Reversed transcriptase, in the following called RT, is a critical enzyme which is responsable for the synthesis of DNA from viral RNA for all retroviruses, including human immune deficiency virus (HIV). Three different enzymatic activities are involved in this process: synthesis of the first DNA strand, degradation of the viral RNA strand in the DNA/RNA hybride and synthesis of the second DNA strand. See e.g. S. P. Goff, (1990) Review, J. AIDS 3, pages 817-831 for an overview.
The RNA and DNA dependent polymerase activities are obviously mediated by the same catalytical group, whereas RNas H activity is mediated through a special part of the molecule. Se e.g. J. Hansen et al. EMBO J. (1988) 7, pages 239-243, M. S. Johnson et al. (1986) PNAS 83, pages 7648-7652. HIV 1 RT appears as a heterodimer in virions consisting of two polypeptides, p66 and p51, with the same N terminals [see e.g. M. M. Lightfoot et al. (1986) J. Virol, 60, pages 771-775, Veronese et al. (1986) Science 231, pages 1289-1291]. p51 is generated by viral protease cleavage of the p66-polypeptides in the dimer of p51 and p15 (see e.g. Mous). In the heterodimer p66 catalyses the polyemerase reaction, but the same sequence in p51 does not [see e.g. Le Grice et al. (1991) EMBO J. 10, pages 3905-3911, Hostomsky et al. (1992), Science 256, pages 1783-1790]. It is assumed that p51 is a part of the binding site for the tRNA primer and a part of the template-primer binding site (see e.g. Kohlstaedt 1992). The RNA-ase H peptid activity in the p66/p51 heterodimer is locatized to the C terminal part of p66 (see e.g. Hansen 1988).
Analysis relating to RT activity has become an accepted technic for detectioning and quantification of retrovirus in cell cultures (see e.g. Pioesz 1980 and Barr-Sinoussi 1983). Together with the p24 antigen test it is used as a confirming test for HIV isolation (see e.g. Jackson 1988, Gupta 1987). RT is also the main target in attempts to find efficient anti-virales against HIV. Many attempts have been made to find selective inhibitors of this enzyme. Although no cure for AIDS not yet has been found, a valuable effect is achieved by treating patients with 3'-azido-3'-deoxy thymidine (AZT). It has, however, been found that treatment with AZT only in a rather short period of treatment (from 3 months to 1 year) produces therapy resistant HIV, with mutated RT. RT tests are presently used for evaluation of reaction mechanisms in potential antivirals. Another great potential use of RT analysis is consequently characterisation of enzymes from therapy resistant mutating viruses.
Conventional measurement of RT activity is carried out with the enzyme in solution, an artificial template/primer construct and tritiated deoxynucleoid triphosphate as the nucleotide substrate (see e.g. Baltimore 1971, Moon & Lee). this system is based on detecting incorporation of radio activity in RNA/DNA hybrides which can be precipitated with trichloro acetic acid (TCA). By using .beta. emitting nucleotides scintillation liquids can be used for detecting radio activity, but this often results in poor reproducability depending on extinction problems. The standard test is coparatibly labor consuming and can not readily be adapted to large scale investigation of a large number of samples. It is also very sensitive to the effects of disturbing factors in the enzyme samples. The latter are often the critical factor for the determination of RT activity in extracts from infected cell cultures or HIV infected individuals. Furthermore, the activity of cellular gammapolymerase enzyme is a potential specificity problem.
During recent years the intensive research relating to HIV has resulted in improvements of RT tests by the use of various techniques.
The incorporation of .sup.125 I labelled substrate resulted in improved sensitivity and eliminated quenching and use of scintillation liquids (Gronowitz et al. 1990). The introduction of templates or primers coupled to solid phases simplified the separation between substrate and product, simplified the need of precipitation with TCA and resulted in a "one tube RT test" (see e.g. Gronowitz 1991, Urabe et al. 1992). Several attempts have also been made to make the test systems less sensitive to disturbing factors.
According to Porstmann et al. 1991 a monoclonal antibody against HIV1-RT is used, which binds to a well in a microplate in order to isolate RT before the analysis. However, this method has been used for determining the RT activity in virus samples which already have been purified by precipitation with PEG and subsequently centrifugation. Furthermore, after immobilization of RT, a complex procedure is used for separating the nucleoside substrate and the soluble labled product. Gronowitz et al. 1991 uses prA/odT bound to a plastic carrier, for affinity purification of HIV-RT in one step directly from cell extracts. The same prA/odT construct is then used as primer/template in the subsequent RT test step.
Recently it has been avoided to use radio activity as a marker in RT analysis by instead using modified nucleotide bases containing antigenic epitopes or structures having av high affinity for defined ligands. The presence of these epitopes or structures in the recently synthesize RNA/DNA hybride is then used for binding antibodies or ligands which have been conjugated with Eg ELISA enzymes. The amount of bound ELISA enzymes has then been determined with a secondary enzym test. Porstmann et al., 1991, makes use of 5-bromo-deoxy-uridine (BrdU) triphosphate as the nucleotide substrate in RT analysis. The amount of incorporated BrdU is determined in a second step with an immune test while using monoclonal anti-BrdU antibodies.
Beery & Scieb, 1992 measures the incorporation of dioxygenine-labled dUTP in newly synthesized DNA instead of radioactively labelled dTTP. In order to be able to separate the non-incorporated nucleotides from newly synthesized DNA also biotine-labelled dUTP is added to the reaction mixture. After reversed transcription the newly synthesized double labelled DNA is immobilized on strept-avidine coated ELISA wells and determined photometrical by binding of peroxydase-conjugated anti-digoxygenine antibodies. This method has been the basis for an RT test kit which is available from Boehringer, Mannheim.
Urabe et al. has developed a non-radioactive RT test which is based on the incorporation of biotine-dUTP in an immobilized odT/prA construct. The amount of incorporated nucleotide substrate is measured photometrical after addition of strept-avidine conjugated alkaline phosphatase (AP).
Another commersially used principle for showing RT activity is to use a series of specific sonds for detecting newly synthesized cDNA. This enzymatic reaction makes use of a heteropolymer RNA molecule with a 20 bases oligonucleotide-primer which is complementary with the RNA sequences close to the 5'end. During the RT reaction a complete cDNA strand is produced. After hydrolysis of template-RNa, cDNA is hybridised with two different oligonucleotide sonds, the capture and detection sonds respectively. The capture sond is used for binding cDNA to wells in a microplate. The detection sonds is conjugated to horeradish peroxidase, which results in a colour reaction after washing for removing unused nucleotide substrates and free sonds.
One purpose of the present invention is to provide a method for quantitative determination of certain polymerase activities which removes most of drawbacks of the previously known methods. Another purpose is to provide a kit for specific determination of different polymerase activities. The invention also relates to the use of said methods and products for novel purposes.
These and other objects of the invention, and how they are achieved, will be explained and illustrated in further detail in the subsequent description and the working examples.
The invention is especially illustrated in connection with determination of HIV1 RT- and Herpes Simplex DNA-polymerase activity, but it can also be used in all highly processive polymerases such as retro RT, DNA virus polymerases, cellular gamma- and delta-polymerases and the like.